The present invention relates to methods and apparatus for the separation of one or more cell fractions from their suspending fluid and/or the resuspension of cells in fresh suspending fluid media. More particularly, the invention relates to automated methods and apparatus that allow for the separation of multiple units of blood simultaneously where the red blood cells and platelet cells are separated from the plasma, the red blood cells are subsequently resuspended in a storage solution, and the platelets are suspended in a concentrating volume of plasma. The method and apparatus dramatically decrease the labor and time required to separate blood into its components and simplifies the data retention required to validate the processing parameters for each unit of blood as required by the evolving FDA regulations governing the safety of the nation""s blood supply. Other embodiments of the invention include in-line filter elements that remove contaminating cells, called leukocytes, which are believed to be responsible for a variety of adverse reactions by the recipient of the blood components. Similarly, other types of filters and packed columns positioned in-line with the flow of these blood components can remove viruses, bacteria or other contaminants, which further enhances the purity and safety of the blood components.
Approximately 12 million units of blood are collected annually in the United States. Another 8 million are collected in the rest of the world. Each donated unit of blood is referred to as xe2x80x9cwhole blood.xe2x80x9d Whole blood contains red blood cells, white blood cells and platelets suspended in a proteinaceous fluid called plasma. Because patients often do not require all of the components of whole blood, most units of whole blood are separated into their multiple components. Individual components are then transfused to different individuals with different needs, a practice referred to as xe2x80x9cblood component therapyxe2x80x9d.
Red blood cells carry oxygen and usually are used to treat patients with anemia. For example, patients with chronic anemia resulting from disorders such as kidney failure, malignancies, or gastrointestinal bleeding and those with acute blood loss resulting from trauma or surgery. White blood cells are responsible for protecting the body from invasion by foreign substances such as bacteria, fungi and viruses.
Plasma contains albumin, fibrinogen, globulins and other clotting proteins. Albumin is a chief protein constituent, fibrinogen plays an important role in the clotting of blood and globulins include antibodies. Thus, plasma serves many functions, including maintenance of satisfactory blood pressures and volume, the control of bleeding by blood clotting, immunity and maintenance of a proper balance of vital minerals in the body. Plasma typically is transfused to control bleeding due to low levels of some clotting factors or it may be transfused to expand the volume of circulating blood. Plasma also may be further fractionated to derive its component proteins.
Platelets help the clotting process by sticking to the lining of blood vessels. Platelets are generally used to improve wound healing and stop bleeding, for example, in patients with leukemia and other forms of cancer.
Cryoprecipitated Antihemophilic Factor (AHF) is rich in certain clotting factors, including Factor VIII, fibrinogen, von Willebrand factor and Factor XIII. It is used to prevent or control bleeding in individuals with hemophilia and von Willebrand""s disease, which are common, inherited major coagulation abnormalities.
Whole blood will separate into its components if treated to prevent clotting and permitted to stand in a container. The red blood cells, weighing the most, will settle to the bottom, the plasma will stay on top, and the white blood cells and platelets will remain suspended between the plasma and the red blood cells. Typically, a centrifuge process is used to speed up this separation.
A common centrifuge process is described in the AABB Technical Manual, methods 9.4 and 9.11 as follows: Typically, the bag of whole blood is carefully loaded into one of the buckets of a large swinging bucket centrifuge. The opposing buckets are weighed and balanced so that their weight is within a few grams. Then, the buckets are loaded into a rotor and the rotor spun at conditions called xe2x80x9clight spinxe2x80x9d by the blood banking community (2000 g for 3 min).
After a considerable wait for the centrifuge to slowly decelerate to zero speed, each bucket is very carefully removed from the rotor so that the bags can be removed from the buckets. This delicate operation must be done in a way that does not disturb or in any way re-suspend the cells. The bag is placed between the two expressing plates of a plasma extractor which force the platelet-rich plasma (PRP) from the whole blood bag to the platelet storage bag. A bag of nutrient solution then is emptied into the packaged red cell bag which is, in turn, placed in storage. The platelet-rich plasma (PRP) can be used to prepare platelets and plasma or Cryoprecipitated AHF.
To make platelets, the platelet-rich plasma (PRP) bags again are balanced and then placed back in the centrifuge for a xe2x80x9cheavy spinxe2x80x9d (5000 g for 5 minutes) causing the platelets to settle at the bottom of the bag. Plasma and platelets then are separated and made available for transfusion. A plasma extractor generally is used to remove all but 50 to 70 ml of plasma, which is required to maintain viability of the platelets. The plasma also may be pooled with plasma from other donors and further processed, or fractionated to provide purified plasma proteins such as albumin, immunoglobulin and clotting factors. Cryoprecipitated AHF may be made from fresh frozen plasma by freezing and then slowly thawing the plasma.
In each case, the components must each be identified in inventory by a method that allows for the traceablilty of that component back to the test results for the original donor, the donated unit, the disposable set in which it was collected, the centrifuge in which it was processed, and, if applicable, the leuko-filter that was used. This traceability is required by law.
Although the centrifuge process speeds up separation of the whole blood into its components, the process is labor intensive and prone to errors and even the most sophisticated inventory control system is subject to the possibility of error as hundreds of data entries are input manually for each unit.
A method and apparatus for the separation of whole blood that is quick, easy and less prone to errors still is needed.
The present invention provides an improved method and apparatus for the separation of whole blood into its components. The method and apparatus automates the separation process, thereby dramatically reducing the labor involved in conventional separation of whole blood. Further, the method and apparatus allows for the separation of multiple units simultaneously, thereby dramatically reducing separation time.
In a preferred embodiment of the present invention, the apparatus includes a centrifuge designed for holding, on a hollow central drive shaft, a plurality of circular cassettes stacked in a co-axial configuration. Each circular cassette has a plurality of cavities for holding a plurality of bags, e.g. a whole blood bag and blood component bags including, for example, a red blood cell bag, a platelet concentrate bag and a platelet poor plasma bag. The cassettes may include further cavities for holding additional components such as filters, other storage bags and an expressor chamber or expressor bag. The various bags are in fluid communication with each other by, for example, tubing or the like to allow transfer of components from one bag to the other. The co-axial configuration is advantageous in that it is self-balancing as the components move from one compartment to another.
Preferably, the whole blood bag and blood component bags are fabricated of materials that allows them to expand and contract repeatedly to move fluids between the cavities. Such materials may include, for example, flexible plastics and elastomeric materials. The number of blood component bags, like the number of cavities, is not limited. The bags for holding the whole blood and blood components are sterile bags fabricated of materials that are of the kind generally approved and accepted for that purpose. Preferably, these bags are shaped to fit the shape of the cassette cavities into which they are placed. Valves and sensors are preferably included in the device to detect and control the flow of the components into the appropriate blood component bag.
In one embodiment, two different types of valves are used. First, an electronic solenoid-driven or motor-driven valve can be used to pinch the tubing connecting the whole blood bag to the red blood cell bag to stop the flow of plasma from being expressed from the whole blood bag as soon as red cells are optically detected in the stream, thereby signaling the end of the expression step. Both the optic detector and the solenoid valve can be controlled by a microprocessor-based logic controller, preferably co-located in the hollow central drive shaft of the device. Power for the optic detector and the solenoid valve can be fed into the rotating housing through a set of concentric slip rings. There is a practical limit on the number of separate power and signal lines that can be fed into the cassette. Therefore, a second type of valve is preferably used that does not require either power or signal communication to the controllers outside the rotating field. This second type of valve could be a mechanical pinch valve, centrifugally actuated, that would open and close based on the speed of the cassette.
In a preferred embodiment, the stacked co-axial configuration operates as follows: a unit of whole blood is collected in a sterile whole blood bag. This whole blood bag is then connected to a sterile bag set via a sterile connection device. This bag set consists of the bags, tubing, and solutions necessary to separate the unit of whole blood into the desired components. These bags are then positioned in the cassettes in the appropriate cavities. The cassettes are closed and loaded into the centrifuge. Under centrifugal force, the red blood cells sediment radially outward in the whole blood bag. After complete sedimentation, expressor fluid or gas is pumped into the expressor chamber or bag, thereby expanding the flexible membrane or bag that contacts the whole blood bag, which compresses the whole blood bag and forces the supernatant fluid (platelet rich plasma) through the platelet concentrate bag and into the platelet poor plasma collection bag. The expressor fluid or gas can have a density higher than that of blood or lower, including air or other suitable gases. During the routing through the platelet concentrate bag, the platelets sediment to the outer surface of the platelet concentrate bag and are collected. This expression continues until all of the supernatant has been expressed from the whole blood bag and an optical sensor detects the presence of red blood cells in the plasma stream. The valves are then closed and the expressor pump stopped. The centrifuge is then stopped and the cassette removed and opened. The bags can then be separated and placed in the appropriate storage containers.
In alternate embodiments, secondary separation devices, such as, filters, or packed columns, etc., are positioned in-line between the product bags in a manner that allows for the removal of target cells as they move from one bag to another while the cassette is spinning and under the influence of the centrifugal force. Examples of this dynamic, in-line secondary separation would include expressing the PRP supernatant through a first leukodepleting filter as it flows from the whole blood bag to the platelet concentrate collection bag.
Another embodiment of additional expression steps can include those required for filters or packed columns or the like that are only effective when operated statically at zero rpm because the centrifugal force interferes with separation performance. For the PRP example above, the separation steps could be modified as follows: after subjecting whole blood to a soft spin, the PRP is expressed into an attached holding bag that can be sealed closed by a valve until the centrifugal speed is zero. Then, the PRP can be expressed through a first leukofilter or other secondary separation means and into the platelet concentrate collection bag. After a hard spin, the platelets will have sedimented and the platelet-poor plasma can be expressed into the plasma bag.
Another embodiment of static separation is the expression of a storage solution into the red blood cell mass to dilute the red blood cells before expressing the mixture through a leukodepleting filter, all occurring a zero speed.
Other embodiments would preferably use an additional processing step. For example, in one embodiment, red blood cells are first obtained by use of, for example, any of the embodiments set out herein. The red blood cells can then be resuspended in other processing chemicals such as, for example, glycerol, which is used for cryopreservation. The reprocessed red cells can later be passed through a column to remove these processing chemicals. Preferably, a series of washes with special solutions will be used to remove most of the offending process chemicals, and the filter or column will remove the residual traces that remain.
Although it can be advantageous to perform these separation steps in the same centrifuge, it may result in unacceptably long processing times in some cases. Thus, in some embodiments, the secondary separation step takes place outside the centrifuge. Preferably, where the secondary separation step occurs outside the centrifuge, the device further includes a built-in refrigerated chamber for controlling the temperature of the cells during the filtering process.
In some embodiments, other fluids, such as sucrose-based storage solutions that are commonly added to separated blood components, are included in the device through the addition of extra bags and cavities. These bags containing, for example, storage solutions, are in fluid communication with the appropriate blood component bag(s) such that, for example, after the blood components have been separated and collected in the appropriate blood component bag(s), the storage solution can be added to the appropriate blood component bag(s). The number of bags and cavities is limited only by the space available in the centrifuge and the space for flow streams within the cassette.
In accordance with another embodiment of the present invention, a radial segment configuration is utilized. In this configuration, a large rotating drum (xe2x80x9crotorxe2x80x9d) is divided into pie-shaped segments, each housing a removable cassette comprised of multiple sections. A bag containing the whole blood is placed in one section of the cassette. The remaining sections of the cassette are used for the containment of the separated blood components. For example, in one embodiment, the cassette consists of three segments, wherein the inner segment contains a first expressor chamber, the middle segment contains both a second expressor chamber and a whole blood bag and the outer segment contains a platelet collection bag. A final plasma collection bag can be positioned on an inside surface of the inner segment. Preferably, a pumping device is used to assist in moving fluid and components from one bag to another.
Preferably, an auto-balancing mechanism, which automatically compensates for the changing state of imbalance of the rotor, is connected to the rotor, thereby eliminating the need for additional balancing steps during the separation process.
In yet another embodiment, the bag arrangements presented previously are shaped to fit into a large swinging-bucket rotor. Swinging-bucket rotors have become common in blood component labs and, thus, this configuration would appeal to the market because labs could use the existing installed base of centrifuges for the process and apparatus of the present invention. Modifications could be made to the rotor and the machine to, for example, allow for expressing fluid to enter the bucket and to position valves and optic detectors on the rotor.
Both the radial configuration and the swinging bucket configuration are used in a manner similar to that described above relating to the stacked disk configuration.